Summary Under this project, a means for the creation of fluorescent nanodiamond (FND) of multiple colors for multiplexed detection in biological assays will be developed. Fluorescent imaging is an ever increasingly important technique in biomedical research with a growing demand for fluorescent labels which are non-toxic, bright, photostable, and which can be easily derivatized with targeting ligands. Nano-sized crystallographic diamond can be made to contain fluorescent color centers by the appropriate processing of atomic lattice constituents to create complexes of impurity atoms and lattice vacancies. Because these created fluorescent centers are embedded in the diamond lattice, they are not susceptible to photobleaching as occurs with other conventional fluorophores. Although red FND has been commercially developed, there have been no means to create bright reproducible FND of other colors. By adopting the method of rapid thermal annealing and using synthetic diamond particles with reproducible nitrogen content and positioning within crystallographic lattice, processing conditions may be achieved to controllably produce color centers unattainable in a conventional thermal annealing process. The colors to be produced will be blue, green, and red, benchmarked for brightness against current commercial organic fluorophors. Each color may be excited by the same ultraviolet wavelength, thereby improving the workflow of multi-signal labeling. Alternatively, all colors are accessible via two-photon absorption of wavelengths from the range 800-1080 nm, allowing for increased tissue penetration and reduced background fluorescence. Due to exceptionally high biological compatibility and unique optical properties, multiplexing via FND has broad applications in immunological assays, flow cytometry, correlative microscopy using multiple fiducial markers, labels for tissue scaffold engineering, and for tagging stem cells in regenerative medicine. In this project, specific utility will be demonstrated using recently developed targeting ligands specific either for vascular endothelial growth factor receptors 1 or 2, which represent potential targets for anti-angiogenic anti-cancer therapies. The outcome of this research will be the demonstration of non-toxic, infinitely photostable fluorescent diamond particles for use in cancer diagnostics.